Abstract
Appropriate amino acid substitutions are critical for protein engineering to redesign catalytic properties of industrially important enzymes like lipases. The present study aimed for improving the environmental stability of lipase from Pseudomonas plecoglossicida S7 through site-directed mutagenesis driven by computational studies. lipA gene was amplified and sequenced. Both wild type (WT) and mutant type (MT) lipase genes were expressed into the pET SUMO system. The expressed proteins were purified and characterized for pH and thermostability. The lipase gene belonged to subfamily I.1 lipase. Molecular dynamics revealed that Y12F-palmitic acid complex had a greater binding affinity (-6.3 Kcal/mol) than WT (-6.0 Kcal/mol) complex. Interestingly, MDS showed that the binding affinity of WT-complex (-130.314 ± 15.11 KJ/mol) was more than mutant complex (-108.405 ± 69.376 KJ/mol) with a marked increase in the electrostatic energy of mutant (-26.969 ± 12.646 KJ/mol) as compared to WT (-15.082 ± 13.802 KJ/mol). Y12F mutant yielded 1.27 folds increase in lipase activity at 55 °C as compared to the purified WT protein. Also, Y12F mutant showed increased activity (~ 1.2 folds each) at both pH 6 and 10. P. plecoglossicida S7. Y12F mutation altered the kinetic parameters of MT (Km- 1.38 mM, Vmax- 22.32 µM/min) as compared to WT (Km- 1.52 mM, Vmax- 29.76 µM/min) thus increasing the binding affinity of mutant lipase. Y12F mutant lipase with better pH and thermal stability can be used in biocatalysis.
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The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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The authors are grateful to ICAR-NBAIM, Mau for financial assistance and infrastructural facilities provided during the work. This is a part of Ph.D. work of the first author under supervision of the corresponding author as co-supervisor.
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HC conceptualized the study, edited and revised the manuscript. PC executed all experimental part along with protein modeling, docking studies, and prepared the first draft of the manuscript. MW, SK and NS performed the Molecular dynamic simulation experiments and analysis. HC, SK, and SS finalized the manuscript in its final form.
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Choudhary, P., Waseem, M., Kumar, S. et al. Y12F mutation in Pseudomonas plecoglossicida S7 lipase enhances its thermal and pH stability for industrial applications: a combination of in silico and in vitro study. World J Microbiol Biotechnol 39, 75 (2023). https://doi.org/10.1007/s11274-023-03518-2
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DOI: https://doi.org/10.1007/s11274-023-03518-2